Nano antibody targeting cadherin 17 and application thereof

Abstract

The invention relates to a nano antibody targeting cadherin 17 and application thereof, and relates to the technical field of immunology and molecular biology, the amino acid sequence of a complementary determining region of the nano antibody targeting cadherin 17 comprises CDR1 shown as any one of SEQ ID NO: 21-32, CDR2 shown as any one of SEQ ID NO: 34-45, and CDR3 shown as any one of SEQ ID NO: 47-58. According to the invention, an alpaca immune phage display library is constructed, a series of nano antibody molecules targeting cadherin 17 (CDH17) are screened from the alpaca immune phage display library, and functions and applications of the nano antibody are verified at various levels of CDH17 antigen protein, CDH17 target cells, CDH17 CAR-T cells and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of immunology and molecular biology, and in particular to a nano antibody targeting cadherin 17 and an application thereof. Background Art

[0002] Cadherins are a superfamily of calcium-dependent cell adhesion molecules that form connections with the cadherin molecules of adjacent cells through their extracellular domains, thereby achieving cell-to-cell adhesion and playing a key role in maintaining tissue structure and morphology. Cadherin-17 (CDH17), also known as liver intestine-cadherin (LI-cadherin) or human peptide transporter-1 (HPT-1), is a non-classical member of the cadherin superfamily. Unlike classical cadherins, the extracellular domain of CDH17 has seven repeating structural units (EC1-EC7) (e.g. Figure 1 The intracellular domain of CDH17 contains only 24 amino acid residues and lacks homology with the intracellular domain of classical cadherins, which consists of 150 to 160 amino acids.

[0003] Current research has proven that CDH17 is an effective and safe target for targeted therapy of digestive system tumors. Candidate antibody molecules against CDH17 can be used to develop diagnostic reagents, targeted antibody drugs and CAR-T / NK immune cell drugs for diseases such as digestive system tumors. However, the currently available candidate antibody molecules against CDH17 are very limited, and they are mainly traditional antibodies. There is only one report of nano antibodies with outstanding advantages and good drugability, which comes from a research team at the University of Pennsylvania and was developed by Chimeric Therapeutics. The world's first CDH17 CAR-T is currently in clinical phase II. In order to promote the research and development of CDH17 target diagnostic reagents and targeted drugs, it is necessary to accelerate the development of more candidate nano antibody molecules against CDH17. In view of this, the present invention discloses nano antibodies targeting cadherin 17 and their applications. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a nanobody targeting cadherin 17 and its application.

[0005] The technical solution of the present invention to solve the above technical problems is as follows:

[0006] In the first aspect, a nanobody targeting cadherin 17, wherein the amino acid sequence of the complementary determining region of the nanobody targeting cadherin 17 includes a CDR1 as shown in any one of SEQ ID NOs: 21 to 32, a CDR2 as shown in any one of SEQ ID NOs: 34 to 45, and a CDR3 with an amino acid sequence as shown in SEQ ID NOs: 47 to 58.

[0007] Further, a nanobody targeting cadherin 17, wherein the nanobody targeting cadherin 17 includes any one of E001, E005, E042, E046, E101, E114, E116, E119, E121, E201, E242, and E243;

[0008] The complementary determining regions of the amino acid sequence of E001 include CDR1 as shown in SEQ ID NO:21, CDR2 as shown in SEQ ID NO:34, and CDR3 as shown in SEQ ID NO:47;

[0009] The complementary determining regions of the amino acid sequence of E005 include CDR1 as shown in SEQ ID NO:22, CDR2 as shown in SEQ ID NO:35, and CDR3 as shown in SEQ ID NO:48;

[0010] The complementary determining regions of the amino acid sequence of E042 include CDR1 as shown in SEQ ID NO:23, CDR2 as shown in SEQ ID NO:36, and CDR3 as shown in SEQ ID NO:49;

[0011] The complementary determining regions of the amino acid sequence of E046 include CDR1 as shown in SEQ ID NO:24, CDR2 as shown in SEQ ID NO:37, and CDR3 as shown in SEQ ID NO:50;

[0012] The complementary determining regions of the amino acid sequence of E101 include CDR1 as shown in SEQ ID NO:25, CDR2 as shown in SEQ ID NO:38, and CDR3 as shown in SEQ ID NO:51;

[0013] The complementary determining regions of the amino acid sequence of E114 include CDR1 as shown in SEQ ID NO:26, CDR2 as shown in SEQ ID NO:39, and CDR3 as shown in SEQ ID NO:52;

[0014] The complementary determining regions of the amino acid sequence of E116 include CDR1 as shown in SEQ ID NO:27, CDR2 as shown in SEQ ID NO:40, and CDR3 as shown in SEQ ID NO:53;

[0015] The complementary determining regions of the amino acid sequence of E119 include CDR1 as shown in SEQ ID NO:28, CDR2 as shown in SEQ ID NO:41, and CDR3 as shown in SEQ ID NO:54;

[0016] The complementary determining regions of the amino acid sequence of E121 include CDR1 as shown in SEQ ID NO:29, CDR2 as shown in SEQ ID NO:42, and CDR3 as shown in SEQ ID NO:55;

[0017] The complementary determining regions of the amino acid sequence of E201 include CDR1 as shown in SEQ ID NO:30, CDR2 as shown in SEQ ID NO:43, and CDR3 as shown in SEQ ID NO:56;

[0018] The complementary determining regions of the amino acid sequence of E242 include CDR1 as shown in SEQ ID NO:31, CDR2 as shown in SEQ ID NO:44, and CDR3 as shown in SEQ ID NO:57;

[0019] The complementary determining regions of the amino acid sequence of E243 include CDR1 as shown in SEQ ID NO:32, CDR2 as shown in SEQ ID NO:45, and CDR3 as shown in SEQ ID NO:58.

[0020] Furthermore, the amino acid sequence of the nanobody targeting cadherin 17 is shown in any one of SEQ ID NOs: 9 to 20.

[0021] In the second aspect, a CAR-T cell targeting E-cadherin 17, wherein the CAR-T cell expresses a chimeric antigen receptor (CAR) targeting E-cadherin 17; the chimeric antigen receptor targeting E-cadherin 17 includes a CDH17 antigen binding domain, a hinge region, a transmembrane region and an intracellular signaling domain; the CDH17 antigen binding domain includes a signal peptide and any one of the nanoantibodies as described in claim 1 or 2.

[0022] Furthermore, the signal peptide is a signal peptide from CD8α, the hinge region is the hinge region of IgG4 with optimized amino acid mutations, the transmembrane region is the transmembrane region of CD28, and the intracellular signaling domain includes the CD28 intracellular co-stimulatory domain, the 4-1BB co-stimulatory domain and the CD3ζ signaling domain.

[0023] Furthermore, the amino acid sequence of the signal peptide of CD8α is shown in SEQ ID NO:60;

[0024] The amino acid sequence of the hinge region of the amino acid mutation-optimized IgG4 is shown in SEQ ID NO: 62;

[0025] The amino acid sequence of the transmembrane region of CD28 is shown in SEQ ID NO:64;

[0026] The amino acid sequence of the CD28 intracellular co-stimulatory domain is shown in SEQ ID NO: 66;

[0027] The amino acid sequence of the 4-1BB costimulatory domain sequence is shown in SEQ ID NO: 68;

[0028] The amino acid sequence of the CD3ζ signaling domain is shown in SEQ ID NO:70.

[0029] In the third aspect, a nucleic acid comprises a nucleic acid sequence encoding the nanobody targeting cadherin 17 or its complementary sequence, or comprises a nucleic acid sequence encoding the chimeric antigen receptor targeting cadherin 17 or its complementary sequence.

[0030] Furthermore, the coding nucleotide sequence of the signal peptide of CD8α is shown in SEQ ID NO:61;

[0031] The nucleotide sequence encoding the anti-CDH17 nanobody is shown in SEQ ID NOs: 82 to 93 or SEQ ID NOs: 74 to 81;

[0032] The nucleotide sequence encoding the hinge region of the amino acid mutation-optimized IgG4 is shown in SEQ ID NO: 63;

[0033] The nucleotide sequence encoding the transmembrane region of CD28 is shown in SEQ ID NO: 65;

[0034] The coding nucleotide sequence of the CD28 intracellular co-stimulatory domain is shown in SEQ ID NO: 67;

[0035] The encoding nucleotide sequence of the 4-1BB costimulatory domain sequence is shown in SEQ ID NO: 69;

[0036] The encoding nucleotide sequence of the CD3ζ signaling domain is shown in SEQ ID NO:71.

[0037] A fourth aspect provides a lentiviral vector, comprising the nucleic acid molecule.

[0038] In a fifth aspect, a pharmaceutical composition is provided, comprising the nano-antibody targeting E-cadherin 17, or the CAR-T cell targeting E-cadherin 17, or the nucleic acid, or the lentiviral vector. Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0039] In the sixth aspect, a nano-antibody targeting E-cadherin 17, or a CAR-T cell targeting E-cadherin 17, or a nucleic acid, a lentiviral vector, or a pharmaceutical composition is used in the preparation of a detection reagent, a living imaging probe, or a therapeutic product targeting E-cadherin 17.

[0040] The core innovation of the present invention is that it provides a group of 12 nano-antibody molecules targeting CDH17. No antibodies with the same CDR1-3 sequences as the present invention have been found in the prior art, which solves the problem of the lack of candidate nano-antibody molecules targeting CDH17 in the prior art.

[0041] The present invention constructs an alpaca immune phage display library, from which a series of 12 nano-antibody molecules targeting cadherin 17 (CDH17) are screened, and the functions and applications of the nano-antibodies are verified at multiple levels such as CDH17 antigen protein, CDH17 target cells and CDH17 CAR-T cells. The fusion antibody (VHH-Fc) of the 12 nano-antibodies and the Fc segment of human IgG1 has good binding activity with human CDH17 antigen protein and gastric cancer cell SNU-16 and pancreatic cancer cell ASPC1 expressing human CDH17 antigen protein, and has cross-binding activity with mouse CDH17 antigen protein. The results of biomembrane interferometry (BLI) technology detection show that the affinity (KD value) of the VHH-Fc fusion antibody of the 12 nano-antibodies to the human CDH17 protein reaches the level of 100pM to nM. Among them, 8 CAR-T cells expressing the amino acid sequences of nano antibody clones E042, E046, E101, E116, E119, E121, E201 and E243 respectively have significant killing ability against gastric cancer and pancreatic cancer cells positive for human CDH17 antigen. The nano antibodies of the present invention can be used for the development of tumor diagnostic reagents, antibody drugs for tumor targeted therapy, and immune cell drugs such as CAR-T / NK.

[0042] The beneficial effects of the present invention are:

[0043] (1) The CDH17 target selected by the present invention is the potential safest and most effective target for the treatment of digestive system tumors: CDH17 is mainly expressed in the tight junctions between epithelial tissue cells of the gastrointestinal system, and is highly expressed in digestive system tumors such as gastric cancer, bile duct cancer, pancreatic cancer, esophageal cancer, neuroendocrine tumors and colorectal cancer cells; existing studies have shown that CDH17 CAR-T or bispecific antibodies do not damage normal expressing tissues, and specifically kill CDH17 expressing tumor tissues.

[0044] (2) The present invention provides candidate nano-antibody molecules for the targeted treatment of digestive system tumors: Compared with blood tumors, the research and development of specific immune targeted therapeutic drugs for solid tumors has been slow, and one of the limiting factors is the lack of effective and safe targets. Current studies have shown that CDH17 is a safe and effective therapeutic target for digestive system tumors, but the research on targeted drugs for CDH17 started relatively late. Currently, there are only a few drugs under development. The bottleneck is in the antibody discovery stage. Only a few CDH17 candidate antibody molecules have been developed, and the nano-antibodies involved are even rarer.

[0045] (3) The nano-antibodies of the present invention have a wide range of applications: (a) The series of candidate nano-antibodies for CDH17 obtained by screening the alpaca immune library of the present invention, the C-terminus of which is fused with the Fc fragment of human IgG1, and the binding activity with the CDH17 antigen is detected at the protein level and the cell level. The results show better or at least equivalent activity than the control antibody. (b) The CAR-T cells constructed using the series of candidate nano-antibody sequences of the present invention have proficient killing activity against a variety of tumor target cells in the digestive system. (c) Nano-antibodies have significant advantages over other antibodies, such as their half-life can be adjusted by chemical modification or protein fusion transformation, strong permeability, can recognize hidden epitopes that are inaccessible to ordinary antibodies, are resistant to pepsin, acid, heat, and easy to produce, and because they are single-chain, they are easy to assemble with other types of antibodies into bivalent antibodies and multivalent antibodies. Therefore, the nano-antibodies of the present invention can be developed into immune detection reagents, CAR-T / NK immune cell drugs, and antibody drugs. Combined with the characteristics of the CDH17 target, the candidate antibodies of the present invention will have better effects if used as cancer immunodiagnostic reagents; if developed into cell or antibody immunotherapy drugs, they will have lower toxic side effects and better clinical efficacy, thereby providing patients with more drug options.

[0046] (4) Can be used to develop dual-target drugs: Studies have found that claudin 18.2, like CDH17, is a safe and effective target for the treatment of digestive system tumors. The two are co-expressed in digestive system tumors such as esophageal cancer, gastric cancer, pancreatic cancer, and colorectal cancer. Clinical or preclinical trials have shown that targeting these two targets can specifically kill tumor cells without harming normal tissues. Therefore, the anti-CDH17 candidate nanoantibody of the present invention can be used in combination with CLDN18.2 antibodies to develop anti-CLDN18.2 / CDH17 dual-antibody drugs and dual-target CAR-T drugs, which is expected to enhance the efficacy of monoclonal antibodies or single-target CAR-T / NK and other immune cell drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 This is a schematic diagram of the CDH17 protein structure, where Extracellular is the extracellular region and Cellular is the cytoplasmic region;

[0048] Figure 2 The results of ELISA test on the titer of alpaca serum after 3rd and 4th immunizations, Serum dilution fold is the serum dilution fold;

[0049] Figure 3 The MF1 value of the flow cytometry test of the binding activity of alpaca four immune sera to CDH17 antigen-expressing cells ASPC-1 and SNU-16;

[0050] Figure 4 Flowchart for the construction of CDH17 alpaca immunization nanobody library;

[0051] Figure 5 It is the ELISA test result that all the enriched output sets have cross-binding with three antigens, Human-CDH17-ECD-His, CDH17-EC1-2-His, and Mus-CDH17-ECD-His, at the nanobody level. The abscissa VHH Con. is the nanobody concentration (μg / mL);

[0052] Figure 6 It is the ELISA test result that all the enriched output sets have cross-binding with three antigens, Human-CDH17-ECD-His, CDH17-EC1-2-His, and Mus-CDH17-ECD-His, at the phage level. The horizontal axis PhageTiter is the phage titer (CFU);

[0053] Figure 7 The figure is a comparison of the amino acid sequences of 12 candidate nanobody clones (VHH1 is a positive control);

[0054] Figure 8The ELISA test results of the binding activity of the full-length expressed proteins of Yangshen antibody Anti-CDH17-VHH-Fc and 12 candidate nanobody clones (E001, E005, E042, E046, E101, E114, E116, E119, E121, E201, E242 and E243E) fused with the Fc segment of human IgG1 to the antigens CDH17-EC1-2-His, Mus-CDH17-ECD-His and CDH17-EC1-2-His, respectively. The abscissa Antibody conc. is the concentration of the full-length expressed antibody protein (μg / mL);

[0055] Fig. 9 The results of FACS detection of the binding activity of the full-length expressed proteins fused with the Fc segment of human IgG1 and the Yangshen antibody Anti-CDH17-VHH-Fc and 12 candidate nanobody clones (E001, E005, E042, E046, E101, E114, E116, E119, E121, E201, E242 and E243E) on ASPC-1 and SNU-16 cells, respectively. The abscissa Antibody conc. is the concentration of the full-length expressed antibody protein (nM);

[0056] Fig.10 Schematic diagram of the common CAR structure of the positive control CDH17 CAR and eight CDH17 candidate nanobody clones (E042, E046, E101, E116, E119, E121, E201, and E243);

[0057] Fig.11 Plasmid map of the third-generation lentiviral expression vector pCDH-EF1-Kan used to construct CDH17 CAR lentiviral expression plasmids for eight candidate nanobody clones (E042, E046, E101, E116, E119, E121, E201, and E243) and control antibodies;

[0058] Fig.12 Flow cytometry analysis of CAR positivity of anti-CDH17 CAR-T and positive control CDH17 CAR-T of 8 candidate nanoantibody clones (E042, E046, E101, E116, E119, E121, E201 and E243);

[0059] Fig.13 The results of flow cytometry detection of CDH17 antigen positivity rate of three target cells (AGS, ASPC-1 and SNU5) that are CDH17 antigen positive and GFP protein expression positivity rate after the introduction of GFP fluorescent marker gene;

[0060] Fig.14The figures show the killing efficiency analysis of anti-CDH17 CAR-T of eight candidate nanoantibody clones (E042, E046, E101, E116, E119, E121, E201 and E243) and positive control CDH17 CAR-T against three target cells (AGS, ASPC-1 and SNU5) that are positive for CDH17 antigen and labeled with GFP fluorescence. DETAILED DESCRIPTION

[0061] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific technology or conditions are specified in the embodiments, the technology or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0062] Example

[0063] 1. Alpaca immunization and immune library screening required antigens, antibodies and cell raw materials

[0064] The present invention uses CDH17 antigen protein and CDH17 antigen high-expressing cells as immunogens to immunize alpacas (strain Alpaca) multiple times, construct an alpaca immune library, and screen antibody molecules that specifically bind to the target antigen CDH17. The raw materials such as antigens, cell lines and positive control antibodies used for immunization and screening are all prepared or provided by Sanyou Biotechnology Co., Ltd., and the relevant information is detailed in Table 1. Among them, CDH17-ECD refers to an antigen protein containing all 7 extracellular domains ECD1-7 of the CDH17 antigen, and CDH17-EC1-2 refers to an antigen protein containing only the first two extracellular domains ECD1-2 of the CDH17 antigen.

[0065] Table 1 Raw materials of antigens and cell lines used for immunization and screening and positive control antibodies

[0066]

[0067]

[0068] The amino acid sequence of the Fc tag in the above antigen protein is from hIgG1-CS, i.e., the constant region of the heavy chain of human IgG1, and an amino acid C (cysteine) in the hinge region is mutated to S (serine) to reduce the presence of free C. The amino acid sequence and encoding nucleotide sequence of the Fc are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively. The above amino acid and encoding nucleotide sequences are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively, and the Anti-CDH17-VHH fused to Fc is from the anti-CDH17 VHH1 (anti-CDH17 VHH1) in patent US20210253728A1.

[0069] 2. Alpaca Immunity

[0070] 2.1 Alpaca immunization method

[0071] Alpaca was cross-immunized with Human-CDH17-ECD-Fc antigen and ASPC-1 cells for a total of 4 immunizations, once every two weeks, with 0.5 mg antigen or 1E+07 (scientific counting method, i.e. 1×10 7 ) cells / alpaca. The first antigen immunization was Freund's complete adjuvant; the subsequent immunization was Freund's incomplete adjuvant, and no adjuvant (purchased from Sigma) was added to the cell immunization. The injection method was subcutaneous multiple injections. The immunization scheme for alpacas is shown in Table 2.

[0072] Table 2 Alpaca immunization scheme

[0073]

[0074] 2.2 Immune serum titer detection

[0075] The alpaca sera were collected after the 3rd and 4th immunizations, and the antibody titer was detected by ELISA and FACS, respectively.

[0076] 2.2.1 Serum titer ELISA test

[0077] Serum titer ELISA detection includes the following steps: (1) Human-CDH17-ECD-his antigen (2 μg / mL, 30 μL) is used to coat the plate and incubate at 4°C overnight; (2) The plate is washed 3 times with PBST and blocked with 5% skim milk powder prepared with PBS (purchased from Yili) at room temperature for 2 hours; (3) The plate is washed 3 times with PBST, 30 μL of serum diluted with PBS is added, and incubated at room temperature for 1 hour; (4) The plate is washed 3 times with PBST, and the secondary antibody Goat anti-Llama IgG (H+L) Secondary Antibody, HRP (ThermoFisher, A16060) is added, and incubated at room temperature for 50 minutes; (5) 30 μl TMB (purchased from SurModics) is added for color development, and the reaction is carried out at room temperature for 15-30 minutes; (6) 50 μl TMB color stop solution (purchased from Bio-Tech, P0215) is added to stop the reaction, and the plate is read with an enzyme reader (Molecular Devices)OD450 value.

[0078] The test samples and controls were three immune serum (CDH17-3rd), four immune serum (CDH17-4th), negative control antibody NC (NC-CDH17) and positive control antibody PC. The serum titer was calibrated according to 1.65 times NC, and the titer was qualified if it was greater than 16K. The results showed that after 3 and 4 immunizations, the titer of alpaca immune serum against Human-CDH17-ECD-his had reached 256K, which can be used for library construction. The serum titer ELISA test results are detailed in Tables 3 and Figure 2 .

[0079] Table 3 Immune serum titer ELISA test results

[0080] Sample dilution factor Three immune serum Four immune serum NC PC 1:2K 2.67 1.98 0.05 2.34 1:4K 2.58 1.64 0.06 1.92 1:8K 1.92 1.15 0.05 2.44 1:16K 1.39 0.58 0.06 2.53 1:32K 0.97 0.37 0.05 2.35 1:64k 0.73 0.24 0.05 1.47 1:128k 0.38 0.15 0.05 0.56 1:256K 0.27 0.11 0.06 0.13

[0081] 2.2.2 FACS detection of immune serum binding to cells

[0082] 1E5 cells / well were added to the plate wells, centrifuged and washed with FACS buffer [1×PBS buffer containing 2% FBS (purchased from Gibco) by volume]; 100 μL serum dilution was added to the cells, incubated at 4°C for 60 minutes, centrifuged and washed three times with FACS buffer; 100 μL Goat anti-Llama IgG (H+L), Fluorescein (FITC) Conjugate (Thermo Fisher, A16061) was added to the cells, incubated at 4°C for 30 minutes. The cells were resuspended with FACS buffer; flow cytometry was performed to analyze the mean fluorescence intensity (MF1).

[0083] The test samples were four immune serum (CDH17-4th), negative control non-immune alpaca serum (NC-CDH17), isotype control antibody IgG (purchased from Sanyou, P78791) and positive control antibody (Reference Ab, i.e. Anti-CDH17-VHH-Fc, P76670, purchased from Sanyou).

[0084] The results showed that the four immune serum samples all maintained good binding with SNU-16 and ASPC-1 cells at 20, 80, 320 and 1280 times dilution concentrations. The results are shown in Tables 4, 5 and Figure 3 .

[0085] Table 4 MF1 values ​​detected by FACS of alpaca tetraimmune serum binding to ASPC-1

[0086] Dilution multiple Four immune serum NC-CDH17 IgG(P78791) Reference Ab 1:20 88324 1672 442 2792 1:80 90931 1169 405 2510 1:320 65455 590 419 1704 1:1280 29190 415 436 914

[0087] Table 5 MF1 values ​​detected by FACS of alpaca tetraimmune serum binding to SNU-16

[0088] Sample dilution factor Four immune serum NC-CDH17 IgG(P78791) Reference Ab 1:20 164023 5965 567 45757 1:80 171792 4082 513 27913 1:320 129986 1750 526 8253 1:1280 63651 854 524 2539

[0089] 3. Construction of alpaca immune library

[0090] 3.1 Immune library construction

[0091] 80 mL of blood from an alpaca after 4 immunizations was collected, and peripheral blood mononuclear cells (PBMC) were separated by Ficoll-Paque density gradient separation solution (GE, 17144003S). RNA was extracted from the separated PBMC and reversely transcribed into cDNA by a reverse transcription kit (TaKaRa, 6210A). Since the molecular form of alpaca antibodies is different from that of ordinary antibodies, it does not contain light chains and the heavy chains do not contain CH1. Therefore, two fragments of different sizes were obtained by PCR by designing primers at the front end and CH2 of the VH germline gene, and the smaller target fragment was recovered by cutting the gel; by comparing all the sequences of all V genes and J genes of the VHH antibody, degenerate primers containing NcoI and NotI restriction sites were designed, and then all VHH genes were amplified using the recovered DNA fragment products as templates, and finally the target antibody gene fragment was inserted into the phage display vector by double enzyme digestion and connection, wherein VHH was fused to the GIII gene at the C-terminus. The ligation product was recovered by a recovery kit (Omega, D6492-02), and finally transformed into competent Escherichia coli SS320 (Lucigen, MC1061F) by an electroporator (Bio-Rad, MicroPulser), and coated on a 2-YT solid plate with ampicillin resistance (prepared by 1.5% tryptone, 1% yeast extract, 0.5% NaCl, 1.5% agar, according to mass volume g / mL). The alpaca immune phage display library thus constructed was used to obtain candidate antibodies through a series of screening processes. The above process is as follows Figure 4 shown.

[0092] 3.2 Determination of single domain antibody library capacity

[0093] The number of clones formed by electroporation was calculated based on the number of clones formed on the plate after dilution of 1 μL of bacterial solution, i.e., the library capacity. The results of the library capacity determination are shown in Table 6, indicating that the single domain antibody library meets the requirement that the library capacity is greater than or equal to 1.00×10 8 Quality standard of CFU.

[0094] Table 6 Single domain antibody library electroporation system and library capacity statistics

[0095] Number of electroporations Single electroporation capacity (CFU) Total reservoir capacity (CFU) 2 <![CDATA[3.69×10 8 ]]> <![CDATA[7.38×10 8 ]]>

[0096] 3.3 Analysis of single domain antibody library sequencing results

[0097] The correct insertion rate of the antibody gene was verified by monoclonal sequencing analysis. A total of 80 clones were selected for sequencing, and the successful sequencing results were further analyzed. The effective library capacity was calculated as follows: total library capacity × antibody correct expression rate × unique sequence ratio = effective library capacity. The results are shown in Table 7.

[0098] Table 7 Statistics of single domain antibody library sequencing information

[0099]

[0100] The results showed that the correct insertion rate of the antibody gene sequence was greater than or equal to 80%, the CDR region alignment had a high richness, the empty ratio was less than or equal to 10%, and the correct rate of enzyme cleavage sites was greater than or equal to 90%, which met the quality standards for the construction of the single domain antibody library. The single domain antibody library of the present invention is of qualified quality and can be used for subsequent screening.

[0101] 4. Screening of Alpaca Immune Library

[0102] The constructed alpaca immune library antibody sequence is displayed on the phage surface through phage display technology, and then specific proteins and cells are used as antigen materials. After multiple rounds of screening, the phage-displayed antibodies binding to the antigen are continuously enriched, and the positive clones that specifically bind to the antigen are further screened through ELISA, and the positive clone antibody sequence is obtained by sequencing.

[0103] 4.1 Immune library screening method

[0104] The immune library screening method comprises the following steps: (1) phage preparation: the alpaca immune library or the obtained output set is subjected to a series of steps such as inoculation, auxiliary phage infection, phage expansion, phage precipitation and resuspension to prepare enriched phage and put into the next step of screening. (2) solid phase screening: the antigen is coated on the surface of an immune tube with high adsorption capacity, and then the prepared phage is added to the immune tube for incubation, washing and elution. After three rounds of panning, the specific monoclonal antibody against the antigen is enriched. (3) liquid phase screening: the biotin-labeled antigen is combined with the magnetic beads coupled with streptavidin, and then incubated, washed and eluted with the prepared phage. After three rounds of panning, the specific monoclonal antibody against the antigen is enriched. (4) cell screening: the prepared negatively screened phage and the antigen-overexpressing cells are incubated, washed and eluted. After three rounds of panning, the specific monoclonal antibody against the antigen is enriched.

[0105] The screening methods and results are detailed in Table 8. The results show that there are different degrees of enrichment starting from the second round. The specific data are shown in Table 8. The methods are: a. solid phase, b. liquid phase, c. cell.

[0106] Table 8 Statistics of audition data

[0107]

[0108]

[0109]

[0110] Note: N / A means not detected or not applicable.

[0111] (5) Selection set detection

[0112] Perform ELISA experiment on the selected output set to detect the enrichment effect:

[0113] ① The ELISA detection steps for nanoantibody VHH levels are as follows: add 2 μg / mL antigen (Human-CDH17-ECD-His, CDH17-EC1-2-His or Mus-CDH17-ECD-His) to the wells of a 96-well plate to coat the plate → block with 5% PBSM → add VHH Pool (VHH output set) dilution solution for incubation → add secondary antibody Anti-Flag-HRP for incubation → add TMB for color development → add TMB color development stop solution → measure OD450 value.

[0114] Control antibody in ELISA detection: Nano antibody VHH against irrelevant targets was used as negative control, i.e. NC (VHH). There are two types of positive control antibodies. In the binding detection of human antigens Human-CDH17-ECD-His and CDH17-EC1-2-His, Anti-CDH17-VHH-Fc against human CDH17 was used as positive control; in the binding detection of mouse antigen Mus-CDH17-ECD-His, VHH-Fc antibody against mouse CDH17, i.e. H_PR_A124 (purchased from Sanyou, P80612), was used as positive control.

[0115] ② The ELISA detection steps for phage Phage level are as follows: add 2 μg / mL antigen (Human-CDH17-ECD-His, CDH17-EC1-2-His or Mus-CDH17-ECD-His) to the wells of a 96-well plate to coat the plate → block with 5% PBSM → add Phage Pool diluent for incubation → add secondary antibody Anti-M13-HRP for incubation → add TMB for color development → add TMB color development stop solution → measure OD450 value; blank Phage was used as a negative control, and NC (Phage). At the same time, 1xPBS buffer was used instead of antigen as a blank control (BC) for parallel ELISA detection.

[0116] The results of the selection set test show:

[0117] At the VHH level: all pools (output sets) were cross-enriched with Human-CDH17-ECD-His, CDH17-EC1-2-His, and Mus-CDH17-ECD-His antigens.

[0118] At the Phage level: All pools had cross-enrichment with Human-CDH17-ECD-His, CDH17-EC1-2-His, and Mus-CDH17-ECD-His antigens; all pools had no nonspecific binding with BC.

[0119] The results of ELISA tests on the selection set are shown in Figure 5 (VHH level) and Figure 6 (Phage level)

[0120] (6) Monoclonal screening

[0121] Monoclonal clones are selected from the enriched pool for preliminary screening. The target antigen is coated on the plate, and a series of steps are followed, including coating, blocking, incubation, adding secondary antibodies, color development, termination, and OD value detection. Positive clones are delineated according to a certain background value and sequenced.

[0122] Human-CDH17-ECD-His, CDH17-EC1-2-His, and Mus-CDH17-ECD-His were used for screening, and a total of 1,656 clones were selected from 19 monoclonal plates. Finally, 623 positive clones that cross-bind with Human-CDH17-ECD-His, CDH17-EC1-2-His, and Mus-CDH17-ECD-His were obtained. After sequence analysis, 36 single-domain antibody (also known as nanobody) molecules with unique sequences were obtained, of which 12 antibody clones E001, E005, E042, E046, E101, E114, E116, E119, E121, E201, E242, and E243 have nucleotide sequences shown in SEQ ID NOs: 82 to 93, and the amino acid sequences encoded by them are shown in SEQ ID NOs: 9 to 20; the amino acid sequences were compared and analyzed, and the results are as follows: Figure 7 The VHH amino acid sequences of the 12 nanoantibody clones were analyzed using the Kabat numbering scheme and definition scheme for CDR1-3, and the results are shown in Table 9. Among them, VHH1 is a positive control antibody, which comes from anti-CDH17VHH1 in patent US20210253728A1.

[0123] Table 9 Amino acid sequences of the three complementary determining regions (CDR1-3) of twelve Nanobodies

[0124]

[0125]

[0126] 5. Full-length expression preparation and physical and chemical testing of candidate nanoantibody molecules

[0127] 5.1 Construction of expression plasmid for full-length antibody protein:

[0128] The coding nucleotide sequence of the nanobody was fused with the coding nucleotide sequence of hIgG1-CS (Fc, whose amino acid sequence and coding nucleotide sequence are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively) for whole gene synthesis and constructed into a pcDNA3.4 vector (Invitrogen) to obtain a VHH-Fc fusion protein expression plasmid.

[0129] 5.2 Expression and purification of full-length antibody protein

[0130] The constructed antibody plasmid was transfected into Expi CHO cells (Gibco, A29133) for transient expression. The antibody expression time was 7 days in an expression volume of 2 mL to 10 mL. After the expression, the protein was purified and packaged.

[0131] 5.3 SDS-PAGE identification of full-length antibody protein Methods and steps for SDS-PAGE identification of full-length antibody protein:

[0132] (1) Preparation of purified protein sample solution:

[0133] Preparation of non-reducing sample solution: Mix the purified sample solution, 4×LDS loading buffer (Zeye Biotechnology, ZY6SL1197) and iodoacetamide (Zeye Biotechnology, ZY144) in proportion to ensure that the final concentration of iodoacetamide is 40 mM and the non-reducing sample loading amount is 1 μg. The mixed sample is placed in a 75°C dry bath and heated for 10 min.

[0134] Preparation of reduced sample solution: The purified sample solution, 4×LDS loading buffer and dithiothreitol (DTT) (Zeye Biotechnology, ZY3483) were mixed in proportion to ensure that the final concentration of DTT was 5 mM and the reduced sample loading amount was 2 μg. The mixed sample was placed in a 100°C dry bath and heated for 10 min.

[0135] (2) Electrophoresis: 140 V, 75 min.

[0136] (3) Staining, destaining, and scanning: The gel was stained with Coomassie Brilliant Blue and scanned with an EPSON V550 color scanner after destaining. (4) Purity calculation: ImageJ was used to calculate the purity of the reduced bands or the purity of the reduced heavy chain plus the light chain according to the peak area normalization method.

[0137] System adaptability: The non-reduced band molecular weight of the reference substance IPI (Ipilimumab) is about 150 kDa, and the purity is greater than 90%; the reduced heavy chain molecular weight is about 50 kDa, the light chain molecular weight is about 25 kDa, and the purity of the heavy chain plus the light chain is greater than 90%.

[0138] 5.4 SEC Characterization of Full-Length Antibody Protein

[0139] Method and steps for SEC identification of full-length antibody protein: (1) Mobile phase preparation: Prepare 0.15M PB+NaCl and adjust the pH to 6.0. (2) Sample treatment: Dilute the sample concentration to 0.5 mg / mL. (3) Chromatographic column conditions: Use XBridge BEH (purchased from Waters), SEC 3.5 μm, 7.8×300 mm, column temperature set at 20°C, detection baseline stable. (4) Parameter settings: flow rate set at 0.8 mL / min; sample injection volume set at 20 μL; detection wavelength 280 nm, bandwidth 4 nm, reference wavelength 360 nm, bandwidth 100 nm, peak width (response time) > 0.1 min (2 s response time); slit 4 nm; negative absorbance baseline 100 mAU.

[0140] System suitability criteria: If the purity of the reference product Herceptin (trastuzumab) monomer is greater than 95%, the separation between BSA monomer and dimer is greater than 1.5, and the baseline is stable, the system is considered to have passed the suitability test.

[0141] The results of the physical and chemical properties test of the 12 candidate antibody Fc fusion proteins constructed and expressed above are shown in Table 10:

[0142] Table 10 Summary of the results of physicochemical property tests of Fc fusion proteins of twelve candidate nanobody clones

[0143]

[0144] Notes: 1) “ / ” means not tested. 2) The IPI band size of the reference product detected by SDS-PAGE was correct and the purity was greater than 95%. The purity of the reference product Herceptin monomer detected by SEC was greater than 95%. The separation degree of BSA monomer and dimer was greater than 1.5, and the baseline was stable, which all met the system adaptability.

[0145] 5.5 Affinity kinetics of full-length antibody proteins

[0146] (1) Solution preparation

[0147] Q Buffer solution: Use 1xPBS to prepare a kinetic buffer solution with a final concentration of 0.025% Tween 20 and 0.2% BSA (IgG-FreeProtease-Free).

[0148] R Buffer solution: Prepare a solution containing 10 mM glycine and 150 mM sodium chloride with ultrapure water, and adjust the pH to 2.0 with hydrochloric acid.

[0149] (2) Affinity kinetics testing of full-length antibody protein

[0150] Methods and steps: ① Select the mode: Open the Gator non-labeled biomolecule analyzer (purchased from Probe Life) and related software, and select the Kinetics experimental mode. ② On the machine: The affinity detection process includes equilibrium, capture, equilibrium, binding, dissociation and regeneration. First, after balancing the baseline, use the Protein A probe (purchased from Probe Life) to capture the antibody to be tested. After balancing again, the antibody to be tested binds and dissociates with the gradient diluted antigen, and finally the probe is regenerated. See Table 11 for details.

[0151] Table 11 Affinity kinetics detection process (Gator)

[0152]

[0153]

[0154] (2) Affinity kinetics test results

[0155] The results of affinity test between each antibody sample and antigen using human CDH17-EC1-2-His (P67889) as antigen are shown in Table 12.

[0156] K detected by Gator label-free biomolecule analyzer D The limit is 1E-06M. The experimental results show that the correlation coefficient R of all antibodies in the Global fitting mode 2 All of them are greater than 0.95, which meets the system adaptability requirements and the results are reliable. The affinity levels of the 12 candidate antibody full-length protein samples to the antigen are all at the nM level or 100pM level, and the affinity level of the positive control antibody Anti-CDH17-VHH-Fc to the antigen is at the 100pM level.

[0157] Table 12 Affinity kinetics test results (Gator)

[0158]

[0159] Note: R 2 : Linear fitting constant; Rmax: Maximum response value of curve fitting.

[0160] The results of affinity test between candidate antibody samples and antigens using mouse antigen protein Mus-CDH17-ECD-His (P98752) as antigen are shown in Table 13. The experimental results show that the correlation coefficients R 2 All of them are greater than 0.95, which meets the system adaptability requirements and the results are reliable. The results show that the dissociation trend data of the samples P210390 (E042) and P210434 (E243) cannot be fitted with the antigen within the dissociation time of 180 seconds, and the affinity levels of the remaining 10 samples to the mouse antigen are at the level of 10nM-10pM. The affinity level of the positive control antibody Anti-CDH17-VHH-Fc to the antigen is at the level of 10nM.

[0161] Table 13 Affinity kinetics test results (Gator)

[0162]

[0163]

[0164] Note: 1) R 2 : Linear fitting constant; Rmax: Maximum response value of curve fitting.

[0165] 5.6 ELISA detection of full-length antibody protein

[0166] Method and steps for ELISA detection of full-length antibody protein: (1) Coating: dilute the antigen with 1×PBS to a concentration of 2μg / mL, add 30μL / well to a 96-well ELISA plate, and coat overnight at 4°C. (2) Blocking: wash the plate three times with PBST, add blocking solution (5% PBSM) and block at room temperature for 2h. (3) Incubation: wash the plate, add 1% PBSM diluted sample at 30μL / well, and incubate at room temperature for 60min. (4) Secondary antibody incubation: wash the plate three times with PBST, add secondary antibody, and incubate at room temperature for 50min. (5) Color development: wash the plate three times with PBST, add 30μL TMB (purchased from SurModics) to each well. (6) Termination: add 50μl TMB color stop solution to terminate the reaction, and detect OD at the same time. 450 .

[0167] In the above test, the negative control antibody NC is IPI ( Ipilimumab ), blank control BC was 1% PBSM.

[0168] The results of ELISA tests between each antibody sample and antigen using CDH17-EC1-2-His, Mus-CDH17-ECD-His and CDH17-EC1-2-His as antigens are shown in Figure 8 As shown in Table 14. All 12 full-length antibodies have strong binding activity with CDH17-EC1-2-His and CDH17-EC1-2-His, and also have different degrees of binding activity with Mus-CDH17-ECD-His.

[0169] Table 14 Summary of ELISA EC50 (μg / mL) of the purified candidate antibody clones full-length expressed proteins

[0170]

[0171]

[0172] Note: / represents not measured, N represents no binding, W represents weak binding, and Y represents binding but EC cannot be calculated. 50 value.

[0173] 5.7 FACS detection of full-length antibody protein

[0174] The binding activity of 12 full-length antibody proteins to two antigen-expressing cells, ASPC-1 and SNU-16, was detected by FACS. The method and steps are as follows:

[0175] (1) Cell plating: Transfer cells from the culture flask to a centrifuge tube, centrifuge to remove the supernatant, resuspend in culture medium, count, and adjust the cell density to 1×10 6 cells / mL. Take a 96-well round-bottom plate and use a 100μL pipette to add cells to each well. Centrifuge at 300g / min for 5min. Discard the supernatant.

[0176] (2) Addition of protein supernatant diluent: Dilute the full-length antibody protein sample with FACS buffer (1×PBS buffer containing 2% FBS by volume) into 8 concentration gradient dilutions of 150.000, 37.500, 9.375, 2.3438, 0.5859, 0.1465, 0.0366, and 0.0092 nM. Use a 100 μL 12-channel pipette to add the antibody dilution to the 96-well cell plate, 100 μL per well. Mix well and incubate in a 4°C refrigerator for 1 h.

[0177] (3) Addition of secondary antibody: Dilute the secondary antibody PE labelled anti-Human Fc (purchased from Jackson) 1:200 with FACS buffer, centrifuge the cell culture plate to remove the supernatant, and use a 100μL 12-channel pipette to add 100μL of the secondary antibody dilution to the cell culture plate. Place the cell culture plate in a 4℃ refrigerator and culture for 30min. Centrifuge to remove the supernatant, wash the plate twice with FACS buffer, and resuspend the cells in each well with 120μL of FACS buffer.

[0178] In the above test, the positive control antibody was Anti-CDH17-VHH-Fc (P76673), the isotype control antibody was IgG1 (P93950-1, purchased from Sanyou), the control with only secondary antibody added was marked as "cell sec", and the blank cell control without any antibody added was marked as "cell only".

[0179] (4) Data acquisition: Turn on the flow cytometer (purchased from Beckman), and after the instrument is cleaned, perform FACS binding detection.

[0180] The FACS test results of antibody samples and antigen-expressing cells are as follows Fig. 9 and as shown in Table 15. The results showed that all 12 full-length antibodies had strong binding activity with ASPC-1 and SNU-16.

[0181] Table 15 FACS detection of EC of the purified candidate antibody clones full-length expressed protein 50 Results Summary (nM)

[0182]

[0183]

[0184] 6. Structural design of Anti-CDH17 CARs

[0185] Nine chimeric antigen receptors targeting CDH17 (Anti-CDH17 CARs) were designed, and the general CAR structure diagram is shown in the figure. Fig.10As shown. Anti-CDH17 CARs include a signal peptide (SP) of CD8α, an anti-CDH17 nanobody (anti-CDH17VHH), an IgG4 hinge region (IgG4mH) with optimized amino acid mutations, a CD28 transmembrane region (CD28TM), an intracellular co-stimulatory domain of CD28, a 4-1BB co-stimulatory domain sequence, and a CD3ζ signaling domain. The amino acid sequences of the nanobody VHH expressed by the 8 CARs are respectively from the 8 nanobody clones E042, E046, E101, E116, E119, E121, E201, and E243 of the present invention, and the corresponding CAR names are abbreviated as E042 CAR, E046 CAR, E101 CAR, E116 CAR, E119 CAR, E121CAR, E201 CAR, and E243 CAR. The amino acid and encoding nucleotide sequence of the nanoantibody expressed by the positive control CAR (abbreviated as CDH17 CAR) is the anti-CDH17 VHH1 sequence in patent US20210253728A1.

[0186] The amino acid and encoding nucleotide sequence of SP are shown in SEQ ID NOs:60 to 61, respectively; the amino acid and encoding nucleotide sequence of IgG4mH are shown in SEQ ID NOs:62 to 63, respectively; the amino acid and encoding nucleotide sequence of CD28TM are shown in SEQ ID NOs:64 to 65, respectively; the amino acid and encoding nucleotide sequence of the CD28 intracellular co-stimulatory domain are shown in SEQ ID NOs:66 to 67, respectively; the amino acid and encoding nucleotide sequence of the 4-1BB co-stimulatory domain are shown in SEQ ID NOs:68 to 69, respectively; the amino acid sequence and encoding nucleotide sequence of the CD3ζ signaling domain are shown in SEQ ID NOs:70 to 71, respectively, and the full-length amino acid sequence and encoding nucleotide sequence of CDH17 CAR as a positive control are shown in SEQ ID NOs:72 to 73, respectively.

[0187] 7. Construction of lentiviral expression plasmid for Anti-CDH17 CARs

[0188] (1) Codon optimization and synthesis of the coding nucleotides of Anti-CDH17 VHH: The VHH coding nucleotide sequences of the eight nanoantibody clones were firstly optimized for human codons and then subjected to DNA synthesis. The coding nucleotide sequences of the optimized eight candidate antibody clones E042, E046, E101, E116, E119, E121, E201 and E243 are shown in SEQ ID NOs: 74 to 81, respectively.

[0189] (2) Vector linearization by enzyme digestion: The third-generation lentiviral expression plasmid pCDH-EF1-Kan (purchased from Fenghui Biotechnology, the plasmid map is shown in Fig.11 The vector backbone is shown, and double restriction digestion is performed at the XbaI and EcoRI restriction sites to linearize the vector.

[0190] (3) Seamless cloning: All DNA fragments in the CAR structure amplified by PCR were cloned into the linearized pCDH-EF1-Kan vector backbone using conventional seamless cloning technology to construct an Anti-CDH17CAR expression plasmid containing 8 nanobodies and 1 positive control, called pCDH17 CAR.

[0191] 8. Lentivirus packaging and titer determination

[0192] The conventional four-plasmid system in the field is used for lentiviral packaging, and all four plasmids are kanamycin-resistant. 293T cells (ThermoFisher) are used as lentiviral packaging cells. The ratio of plasmid dosage of lentiviral expression plasmid pCDH17 CAR and three auxiliary plasmids pMDLg / pRRE, pRSV-Rev and pMD2.G (all three plasmids are purchased from Fenghui Bio) to co-transfect 293T cells is 4:2:2:1; for T75 cell culture flasks, the total amount of plasmid is 20ug, and the dosage of the four plasmids is 8.8ug, 4.4ug, 4.4ug, and 2.2ug respectively. The dosage of transfection reagent PEI is 3 times the total amount of the four plasmids; for T75 culture flasks, the dosage of PEI is 60ug (1ug / ul, 60ul). The above plasmid and PEI transfection reagent were added to serum-free medium and mixed, and then the two were mixed and placed for 15 minutes, and then the mixture was added to the T75 culture flask with 293T cells, gently mixed, and cultured in a 37°C, 5% CO2 cell culture incubator for 6 hours. After 6 hours, the fresh medium containing 2% FBS was replaced and culture was continued. The culture supernatant of the lentivirus was collected 48 hours after transfection, and the supernatant was taken after centrifugation (2000rpm, 15min), filtered through a 0.45um filter, and concentrated by ultracentrifugation (25000rpm, 3h). Then, the virus precipitate was resuspended with the corresponding volume of culture medium according to the dilution multiple, aliquoted, and stored at -80°C.

[0193] For the titer determination of CDH17 CAR lentivirus, the lentivirus stock solution or concentrate was serially diluted and transfected into 293T cells. After 72 hours, the transfection efficiency was detected by flow cytometry, and the active titer of the lentivirus was calculated.

[0194] 9. Preparation and culture of CDH17 CAR-T

[0195] The frozen PBMCs were revived and cultured overnight. The suspended PBMCs were centrifuged and resuspended in X-VIVO15 medium (purchased from LONZA) containing 300 IU / mL IL-2 and the cell density was adjusted to 1.5×10 6 / ml, add the activator MACS GMP T Cell TransAct (Milteny Biotec) at a ratio of 17.5:1 (PBMC volume: activator volume) for activation, centrifuge and replace the culture medium (X-VIVO 15 containing 300IU / mL IL-2) after 30 hours, count, add CDH17 CAR lentivirus at MOI=5, pipette and mix, culture in a cell culture incubator at 37°C and 5% CO2 for 16 hours, add an equal volume of culture medium, and continue to culture. Count every two days and maintain the cell density at 0.5-2×10 6 / ml, and continue to culture for 8-14 days (starting from cell activation), during which the CAR expression positive rate of CDH17 CAR-T cells was detected by flow cytometry as follows:

[0196] Take CAR-T cell solution containing 1E6 cells, centrifuge at 300g for 5min, discard the supernatant, add 50μL PBS to resuspend; add 1-3ul CDH17 antigen CDH17 Protein-His Tag (Acro), incubate at room temperature in the dark for 20min; add 500μL PBS, mix, centrifuge at 300g for 5min; discard the supernatant, add 50μL PBS to resuspend; add CD3 antibody Anti-CD3-APC (Biolegend) and secondary antibody PE anti-His Tag (Biolegend), mix, incubate at room temperature in the dark for 20min; add 500μL PBS, mix, centrifuge at 300g for 5min; discard the supernatant, add 200μL PBS to resuspend the cells, and detect on the machine. Analyze the proportion of CAR-positive T cells in the CD3-positive cell population, which is the CAR positive rate of CAR-T cells. In one specific embodiment, the CAR positivity flow cytometry results of 9 CAR-T cells at 9 days after activation (D9) are as follows: Fig.12 and as shown in Table 16.

[0197] Table 16 CAR positive rate of anti-CDH17 CAR-T cells (D9)

[0198]

[0199] 10. In vitro killing function detection of CDH17 CAR-T

[0200] Three target cells were used, namely, the CDH17 antigen naturally expressing cell line gastric adenocarcinoma AGS (purchased from the Shanghai Academy of Sciences Cell Bank), pancreatic cancer ASPC-1 (purchased from the Chinese Academy of Sciences Cell Bank), and gastric cancer SNU-5 (purchased from the Chinese Academy of Sciences Cell Bank) into which the green fluorescent protein (GFP) marker gene was introduced. The flow cytometry results of the expression positive rates of CDH17 antigen and GFP marker protein of the three target cells are shown in Figure 2. Fig.13 express.

[0201] Killing experiment settings: 9 CAR-T cells and control T (MOCK-T) were used as effector cells and co-incubated with 3 target cells, respectively. The effector: target ratio was set to 2:1 (Note: the effector cell is the total number of cells). Three replicate wells were set for each effector-target co-incubation, and a blank control with only target cells was set. (1) Target plating: In a 96-well plate, 100ul of target cells with a cell density of 2E5 / ml were added to each well. S3 real-time live cell imager (purchased from Sartorius) was used to set the green fluorescence imaging parameters of the target cells and cultured overnight at 37°C and 5% CO2. (2) On the second day, 100ul of effector cells with a cell density of 4E5 / ml were added to the corresponding wells, and 100ul of culture medium was added to the blank control with only target cells. S3 real-time living cell imager (purchased from Sartorius) was used to incubate the cells at 37°C and 5% CO2 for 48 h.

[0202] Result analysis: The green fluorescence images of target cells taken in real time by IncuCyte were used for analysis. The most recent shooting time point before the addition of effector cells and the start of co-incubation with target cells was used as the time point for normalizing the target cell fluorescence area. The end point of co-incubation was used as the endpoint to draw the target cell growth curve, that is, the killing curve of effector cells against target cells. The killing efficiency of effector cells was calculated based on the green fluorescence value of target cells corresponding to the end point of co-incubation on the killing curve. The calculation formula is: Killing efficiency = [(blank target cell endpoint fluorescence value - effector-target co-incubation cell group endpoint fluorescence value) / blank target cell endpoint fluorescence value] × 100%. Draw a killing efficiency bar graph based on the killing efficiency calculation results (see Figure 4 ). It can be seen that the 9 CAR-Ts have a significant killing effect on the three CDH17 target cells. Compared with MOCK-T, the difference in killing is between 1*(P<0.05) and 4*(P<0.0001). Among them, the killing efficiency of E119CAR-T and E121 CAR-T is relatively low, which is related to their low CAR positivity rate. Except for E119 CAR-T and E121CAR-T, the CAR-T killing efficiency of the other 6 candidate antibodies is equivalent to or higher than that of the control CAR-T. In addition, the killing effect of the 9 CAR-T on the two gastric cancer cells AGS and SNU-5 is better than that on the pancreatic cancer cell ASPC-1.

[0203] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A nanobody targeting cadherin 17, characterized in that: The amino acid sequence of the complementary determining region of the nanobody targeting cadherin 17 includes a CDR1 as shown in any one of SEQ ID NOs: 21 to 32, a CDR2 as shown in any one of SEQ ID NOs: 34 to 45, and a CDR3 with an amino acid sequence as shown in any one of SEQ ID NOs: 47 to 58.

2. The nanobody targeting cadherin 17 according to claim 1, characterized in that: The amino acid sequence of the nanobody targeting cadherin 17 is shown in any one of SEQ ID NOs: 9 to 20.

3. A CAR-T cell targeting E-cadherin 17, characterized in that: The CAR-T cells express a chimeric antigen receptor targeting E-cadherin 17; the chimeric antigen receptor targeting E-cadherin 17 includes a CDH17 antigen binding domain, a hinge region, a transmembrane region and an intracellular signaling domain; the CDH17 antigen binding domain includes a signal peptide and any one of the nanoantibodies as described in claim 1 or 2.

4. A CAR-T cell targeting E-cadherin 17 according to claim 3, characterized in that: The signal peptide is the signal peptide of CD8α, the hinge region is the hinge region of IgG4 with optimized amino acid mutations, the transmembrane region is the transmembrane region of CD28, and the intracellular signal transduction domain includes the CD28 intracellular co-stimulatory domain, the 4-1BB co-stimulatory domain and the CD3ζ signal transduction domain.

5. A CAR-T cell targeting E-cadherin 17 according to claim 4, characterized in that: The amino acid sequence of the CD8α signal peptide is shown in SEQ ID NO: 60; The amino acid sequence of the hinge region of the amino acid mutation-optimized IgG4 is shown in SEQ ID NO: 62; The amino acid sequence of the transmembrane region of CD28 is shown in SEQ ID NO:64; The amino acid sequence of the CD28 intracellular co-stimulatory domain is shown in SEQ ID NO: 66; The amino acid sequence of the 4-1BB costimulatory domain sequence is shown in SEQ ID NO: 68; The amino acid sequence of the CD3ζ signaling domain is shown in SEQ ID NO:

70.

6. A nucleic acid, characterized in that It comprises a nucleic acid sequence encoding a nanobody targeting cadherin 17 as described in any one of claims 1 to 2 or its complementary sequence, or comprises a nucleic acid sequence encoding a chimeric antigen receptor targeting cadherin 17 as described in any one of claims 3 to 5 or its complementary sequence.

7. A nucleic acid according to claim 6, characterized in that The coding nucleotide sequence of the CD8α signal peptide is shown in SEQ ID NO:61; The coding nucleotide sequence of the anti-CDH17 nanobody is shown in any one of SEQ ID NOs: 82 to 93 or SEQ ID NOs: 74 to 81; The nucleotide sequence encoding the hinge region of the amino acid mutation-optimized IgG4 is shown in SEQ ID NO: 63; The nucleotide sequence encoding the transmembrane region of CD28 is shown in SEQ ID NO: 65; The coding nucleotide sequence of the CD28 intracellular co-stimulatory domain is shown in SEQ ID NO: 67; The encoding nucleotide sequence of the 4-1BB costimulatory domain sequence is shown in SEQ ID NO: 69; The encoding nucleotide sequence of the CD3ζ signaling domain is shown in SEQ ID NO:

71.

8. A lentiviral vector, characterized in that The lentiviral vector comprises the nucleic acid molecule of claim 6 or 7.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the nanobody targeting cadherin 17 as described in any one of claims 1 to 2, or the CAR-T cell targeting cadherin 17 as described in any one of claims 3 to 5, or the nucleic acid as described in any one of claims 6 to 7, or the lentiviral vector as described in claim 8.

10. Use of a nanobody targeting E-cadherin 17 as described in any one of claims 1 to 2, or a CAR-T cell targeting E-cadherin 17 as described in any one of claims 3 to 5, or a nucleic acid as described in any one of claims 6 to 7, or a lentiviral vector as described in claim 8, or a pharmaceutical composition as described in claim 9 in the preparation of a detection reagent, in vivo imaging probe or therapeutic product targeting E-cadherin 17.

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